Recently, a forced-air-ventilated micro-isolation system (FVMIS) has been recognized to accurately maintain microenvironmental conditions inside cages, but the details of the relationship between the concentrations of carbon dioxide (CO2) and oxygen (O2) and the air change rate inside the cages have never been reported. In this study, the proper intra-cage air change rate was examined based on the CO2 concentration and O2 concentration inside the cages measured by changing the ventilation volume inside the closed cages of the FVMIS while housing animals. In the experiments, three 8-week-old Wistar strain male rats weighing 303 g on average were housed in each FVMIS cage (capacity: 0.0223 m3), and the temperature, relative humidity, CO2 concentration and O2 concentration were measured when the air change rate inside the cages was varied from 10 air changes per hour (ACH) to 120 ACH. It proved that the CO2 concentration in the FVMIS cages decreased uniformly with the increase in the air change rate. As a result, 60 ACH was required to maintain the CO2 concentration level inside the FVMIS cages equivalent to or less than that in the conventional housing. Otherwise, when based on the O2 concentration, 50 ACH was required. In consideration of these results and others based on ventilation, airflow, temperature and the ammonia concentration reported previously, we concluded that the proper air change rate inside the FVMIS cages should be approximately 60 ACH.